Synthesis, Characterization and Biological Activities of Zinc Oxide Nanoparticles Derived from Secondary Metabolites of Lentinula edodes.

Zinc oxide nanoparticles (ZnO NPs) are the second most prevalent metal oxide, owing to their characteristics of low cost, safe, and easily prepared. ZnO NPs have been found to exhibit unique properties which show their potential to be used in various therapies. Numerous techniques have been devised for the manufacture of zinc oxide because it is one of the nanomaterials that has received major research interest. Mushroom sources are proven to be efficient, ecologically friendly, inexpensive, and safe for humankind. In the current study, an aqueous fraction of methanolic extract of Lentinula edodes (L. edoes) was used to synthesize ZnO NPs. The biosynthesis of ZnO NPs was achieved by using the reducing and capping capability of an L. edodes aqueous fraction. Bioactive compounds from mushroom, such as flavonoids and polyphenolic compounds, are used in the green synthesis process to biologically reduce metal ions or metal oxides to metal NPs. Biogenically synthesized ZnO NPs were further characterized by using UV-Vis, FTIR, HPLC, XRD, SEM, EDX, zeta sizer and zeta potential analyses. The FTIR showed the functional group at the spectra in the range 3550-3200 cm-1 indicated the presence of the hydroxyl (OH) group, while bands in the range 1720-1706 cm-1 indicated C=O carboxylic stretches bonds. Furthermore, the XRD pattern of ZnO NPs created in the current study was found to be nanocrystals which are hexagonal. The SEM analysis of ZnO NPs showed spherical shapes and size distributions in the range 90-148 nm. Biologically synthesized ZnO NPs have substantial biological activities including antioxidant, antimicrobial, antipyretic, antidiabetic and anti-inflammatory potential. Biological activities showed significant antioxidant (65.7 ± 1.09), antidiabetic (85.18 ± 0.48), and anti-inflammatory potential (86.45 ± 0.60) at 300 µg inhibition in paw inflammation of (1.1 ± 0.06) and yeast-induced pyrexia (97.4 ± 0.51) at 10 mg in a dose-dependent manner. The outcomes of this research indicated that ZnO NPs significantly reduced inflammation and have the ability to scavenge free radicals and prevent protein denaturation, while also indicating their possible use in food and nutraceutical applications to treat various ailments.

Sustained virological response to antiviral drugs in treatment of different genotypes of HCV cirrhotic patients.

Cirrhosis and liver cancer are both caused by hepatitis C virus (HCV) infection of the liver. Patients with HCV cirrhosis may be treated with one of many antiviral medications, depending on their specific genotype. Samples of cirrhotic HCV were obtained from 190 people at the Khyber Teaching Hospital and the Hayatabad Medical Complex in Peshawar, Pakistan. Multiplex and real-time PCR were used to assess the genotypes and viral loads of the samples, respectively. Sixty patients were given sofosbuvir plus daclatasvir with ribavirin, while the remaining 56 patients were given sofosbuvir with ribavirin for a period of 12-24 weeks. LFTs were also tracked both before and after therapy. Group I (sofosbuvir + daclatasvir) had a sustained virological response of 82.70 percent. Group II (sofosbuvir + daclatasvir with ribavirin) had an 86% sustained virological response, whereas group III (84% sustained virological response) received only ribavirin. When compared to other genotypes, genotype 3 showed the most impressive sustained virologic response (SVR) to the antiviral medicines. Based on the results of this trial, we propose sofosbuvir + daclatasvir ribavirin for the treatment of cirrhotic patients with various HCV genotypes since it produces the greatest sustained virological response.

Aspergillus flavus originated pure compound as a potential antibacterial.

PROBLEM BACKGROUND: Penicillin was the first and most famous fungal secondary metabolite used as broad spectrum antibiotic that revolutionarised pharmaceutical research and also saved millions of lives. The over optimistic belief in 1967 that sufficient antibiotics had been discovered to defeat infectious diseases was quickly crashed with the appearance of multidrug resistant (MDR) bacteria in 1990s. This has posed a serious threat to mankind. Although scientists are making efforts to synthesize and discover new antibiotics there are not enough new drugs in pharmaceutical pipeline to beat the pace at which MDR bacteria are emerging. In view of this there is an urgent and serious medical need for new bioactive compounds to be discovered to treat infections caused by MDR pathogens. The present study is aimed to investigate the antibacterial potential of Aspergillus flavus originated compounds that may act as drug leads to treat future infections. METHODOLOGY: Among the 6 isolated fungal strains from the rhizosphere of Mentha piperetta, one was processed for isolation of secondary metabolites on the basis of preliminary antibacterial testing. Observation of morphological and microscopic features helped in identification of the fungal strain as Aspergillus flavus. Potato Dextrose Agar (PDA) medium was used for fungal growth while Czapec Yeast Broth (CYB) medium was used for production of fungal metabolites. Column chromatography technique was utilized for purification of compound from crude fungal extract and the mass of the compound was determined using Liquid Chromatography Mass Spectrometry (LCMS) method. Structure elucidation of the pure compound was performed using 500 Varian Nuclear Magnetic Resonance (NMR) machine. Docking was performed using Glide SP algorithm. Agar well diffusion method was used to determine the invitro antibacterial potential of the compound against two MDR bacterial strains i.e. Staphylococcus aureus and Proteus vulgaris. For this a total of 4 dose concentrations i.e. (100, 250, 500, 1000 µg mL- 1) of the compound were prepared and applied to bacterial strains on Mueller Hinton agar using tetracycline as control. RESULTS: The chemical name of the purified compound from A. flavus was determined as (2E)-3-[(3S, 4R)-8-hydroxy-3, 4-dimethyl-1-oxo-3, 4-dihydro-1H-2- benzopyran-7-yl] prop-2-enoic acid with the formula C14H14O5 and exact mass of 262.08. The in-Silico analysis showed that this compound has the potential to inhibit the binding pocket of S. aureus TyrRS (1JII) with docking score of - 8.67 Kcal mole- 1. The results obtained from invitro experiments were encouraging as at 1000 µg mL- 1 the compound showed 58.8% inhibition against S. aureus and 28% inhibition against P. vulgaris. CONCLUSIONS: The pure compound with formula C14H14O5 and exact mass of 262 exhibited antibacterial potential both insilico and invitro against both Gram negative and Gram positive bacteria. The compound was more active against S. aureus in comparison to P. vulgaris. From the obtained results it is concluded that this compound can be used as potent antibacterial candidate but further studies will be needed prior to its use as antibiotic.

Evaluation of crude saponins, methanolic extract and subsequent fractions from Isodon rugosus Wall. ex Benth: Potentials of anti-angiogenesis in egg and anti-tumorigenesis in potato.

Based on the ethnomedicinal use of Isodon rugosus the current study was designed to evaluate its crude saponins (Ir.Sp), and subsequent fractions for anti-angiogenic and anti-tumor potentials. Chorioallantoic membrane (CAM) assay was used in anti-angiogenic potentials with Dexamethasone as positive control. The antitumor activity was evaluated with potato disk method using Vincristine sulfate as positive control. Moreover, antibacterial activity was also conducted against A. tumefaciens. The highest anti-angiogenic effect was observed with Ir.Sp, i.e., 79.00±0.58% at concentration of 1000 µg/ml which drop drown to 48.67±1.20% at lowest tested concentration of 31.25 µg/ml with IC50 of 41 µg/ml. Similarly, in the anti-tumor activity the Ir. Chf revealed excellent inhibition of tumor with IC50 value of 60 µg/ml. All the samples (excluding Ir. Sp and Ir. Cr) were inactive against A. tumefaciens, which demonstrates that the samples which did not show any antibacterial activity are rich in certain bioactive principles which may be responsible for the anti-tumor and anti-angiogenic potentials. Our results conclude that the Ir.Sp, Ir.Chfmay be good targets for isolation of bioactive compounds responsible for the inhibition of excessive proliferation of cells and angiogenesis.

Conservation of the extended substrate specificity profiles among homologous granzymes across species.

Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologs in mice; however for granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e. beyond cleavage positions P4-P4') match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.

Analysis of the evolution of granule associated serine proteases of immune defence (GASPIDs) suggests a revised nomenclature.

GASPIDs (granule associated serine protease of immune defence) are a family of serine proteases intimately involved with the function of the vertebrate immune system. With the availability of a large and growing set of assembled genomes, we undertook an evolutionary analysis to plot the development of this protein family from a single precursor to the modern mammalian cohort of 12 genes, in an attempt to define and systematically classify subgroups or clades within this family, which are implied by the conventional gene designations. We identified a primordial GASPID gene as either GzmA or GzmK in cartilaginous fish and reconstructed an evolutionary path through to humans. Apart from historic value, the current sub-designations (granzymes, mast cell proteases and neutrophil serine proteases) serve no useful purpose and are increasingly misleading. We therefore used our phylogenetic and point mutation analyses to separate GASPIDs into three clades. These could form the basis of a simple nomenclature that allows effective classification of GASPIDs without implying functional roles.

Purification of andibenin and a chromanone analogue from rhizospheric Aspergillus flavus and soil-borne Penicillium notatum exhibiting cytotoxic and antibacterial properties.

The discovery of bioactive compounds from fungal natural sources holds immense potential for the development of novel therapeutics. The present study investigates the extracts of soil-borne Penicillium notatum and rhizosphere-inhabiting Aspergillus flavus for their antibacterial, antifungal, and cytotoxic potential. Additionally, two compounds were purified using chromatographic and spectroscopic techniques. The results demonstrated that the ethyl acetate fraction of A. flavus exhibited prominent cytotoxic activity against Artemia salina, whereas the ethyl acetate fraction of P. notatum displayed promising antibacterial potential. At dose concentrations of 10, 100, and 1000 µg mL-1, the ethyl acetate fraction of A. flavus showed mortality percentages of 7.6%, 66.4%, and 90%, respectively. The ethyl acetate fraction of P. notatum extract exhibited significant antibacterial activity, forming inhibition zones measuring 41, 38, 34, 34, and 30 mm against B. subtilis, S. flexneri, E. coli, K. pneumoniae, and S. aureus, respectively, at 1000 µg mL-1. At this concentration, inhibition zones of 28, 27, and 15 mm were recorded for P. vulgaris, S. typhi, and X. oryzae. Using bioassay-guided approach, one compound each was purified from the fungal extracts. The initial purification involved mass spectroscopic analysis, followed by structural elucidation using 500 MHz nuclear magnetic resonance (NMR) spectroscopy. Compound 1, derived from A. flavus, was identified as ethyl 2-hydroxy-5,6-dimethyl-4-oxocyclohex-2-ene-1-carboxylate, with a mass of 212, whereas compound 2, isolated from P. notatum, was identified as 3-amino-2-(cyclopenta-2,4-dien-1-ylamino)-8-methoxy-4H-chromen-4-one, with an exact mass of 270. Based on bioassay results, compound 1 was subjected to brine shrimp lethality assay and compound 2 was tested for its antibacterial potential. Compound 1 exhibited 30% lethality against brine shrimp larvae at a concentration of 100 µg mL-1, whereas at 1000 µg mL-1 the mortality increased to 70%. Compound 2 displayed notable antibacterial potential, forming inhibition zones of 30, 24, 19, and 12 mm against S. aureus, E. coli, B. subtilis, and S. flexneri, respectively. In comparison, the standard antibiotic tetracycline produced inhibition zones of 18, 18, 15, and 10 mm against the respective bacterial strains at the same concentration.

Purification of Potential Antimicrobial Metabolites from Endophytic Fusarium oxysporum Isolated from Myrtus communis.

The rise of microbial resistance and emerging infections pose significant health threats. Natural products from endophytic fungi offer a promising source of novel compounds with the potential as major drug leads. This research aims to screen Myrtus communis and Moringa oleifera for endophytic fungi and screen their metabolites for antibacterial and antifungal potential. Six endophytic fungal strains were isolated using a potato dextrose agar (PDA) medium. The M. communis isolates were designated MC1, MC2, and MC3, and the M. oleifera isolates were named MO1, MO2, and MO3. Preliminary bioactivity testing revealed that the MC3 isolate exhibited significant growth inhibition against multidrug-resistant bacterial and fungal pathogens, including Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Candida glabrata. The MC3 isolate was identified as Fusarium oxysporum through morphological and microscopic methods. For metabolite production, the fungal strain was cultured in potato dextrose broth (PDB) medium at 28 °C for 14 days in a shaking incubator. The metabolites were purified using various chromatographic techniques, HPLC and GC-MS. The GC-MS analysis of the bioactive compound containing fungal strain (F. oxysporum) revealed multiple compounds at different retention times using the NIST-20 Library. Based on RSI values and probability indices, two compounds were targeted for further purification. Structure elucidation was performed using 1D and 2D nuclear magnetic resonance (NMR) experiments on a Varian 500 NMR machine. The compounds identified were ethyl iso-allocholate (C26H44O5, exact mass 436.32) and 1-monolinoleoyl glycerol trimethylsilyl ether (C27H56O4Si2, exact mass 500.37). The MS (NIST-20) library facilitated the investigation of the in silico antimicrobial activity of these compounds against the elastase virulence protein of P. aeruginosa and protease Sapp1p from C. parapsilosis. Both the compounds were docked with druggable proteins using the Glide induced fit docking (IFD) algorithm. The ethyl iso-allocholate and 1-monolinoleoyl glycerol trimethylsilyl ether compounds showed binding scores - 10.07 kcal mol-1 and - 7.47 kcal mol-1 against elastase, and - 8.16 kcal mol-1 and - 6.89 kcal mol-1 against aspartic protease, respectively. In vitro studies confirmed the inhibitory activity of these compounds against multidrug-resistant P. aeruginosa and E. faecalis. Ethyl iso-allocholate exhibited higher bioactivity against P. aeruginosa with inhibition rates of 41%, 27%, and 35% at concentrations of 1000, 500, and 250 µg mL-1, respectively. These results suggest that bioactive compounds from F. oxysporum have the potential as antimicrobial agents, warranting further research.

On multivalued contractions in cone metric spaces without normality.

Wardowski (2011) in this paper for a normal cone metric space (X, d) and for the family A of subsets of X established a new cone metric H : A × A → E and obtained fixed point of set-valued contraction of Nadler type. Further, it is noticed in the work of Jankovic et al., 2011 that the fixed-point problem in the setting of cone metric spaces is appropriate only in the case when the underlying cone is nonnormal. In the present paper we improve Wardowski's result by proving the same without the assumption of normality on cones.

Fixed point results of locally contractive mappings in ordered quasi-partial metric spaces.

Fixed point results for a self-map satisfying locally contractive conditions on a closed ball in an ordered 0-complete quasi-partial metric space have been established. Instead of monotone mapping, the notion of dominated mappings is applied. We have used weaker metric, weaker contractive conditions, and weaker restrictions to obtain unique fixed points. An example is given which shows that how this result can be used when the corresponding results cannot. Our results generalize, extend, and improve several well-known conventional results.

Phylogenetic Analysis and Emerging Drug Resistance against Different Nucleoside Analogues in Hepatitis B Virus Positive Patients.

Several nucleotide analogues have been approved for use in treating hepatitis B virus (HBV) infection. Long-term exposure to therapy leads to the emergence of mutations within the HBV DNA polymerase gene, resulting in drug resistance, a major factor contributing to therapy failure. Chronic HBV patients from the Khyber Pakhtunkhwa province, Pakistan, who had completed 6 months of therapy participated in this study. Samples were collected from 60 patients. In this study, the entire reverse transcriptase domain of the HBV polymerase gene was amplified using nested polymerase chain reaction and sequenced. Drug-resistant mutations were detected in nine (22.5%) patients. All of these patients had lamivudine-resistant mutations (rtM204V + L180M), while seven individuals (17.5%) had both lamivudine- plus entecavir-resistant mutations (L180M + M204V + S202G). N236T, a mutation that gives rise to tenofovir and adefovir resistance, was observed in two (5%) patients. T184A, a partial drug-resistant mutation to entecavir, was found in five (12.5%) patients. Furthermore, other genotypic variants (100%) and vaccine escape mutations (5%) were additionally observed. Moreover, pN459Y (35%), pN131D (20%), pL231S (20%), pP130Q (17.5%), pS189Q (12.5%), pP161S (5%), pH160P (2.5%), pT322S (2.5%), and pA223S (2.5%) mutations in the polymerase gene, as well as sA166V (17.5%), sQ181K (12.5%), sV184R (7.5%), sA17E (5%), sP153S/K (5%), sW156C (5%), sC76Y (2.5%), and S132F (2.5%) mutations in the small surface gene, were identified for the first time in this study. Phylogenetic analysis showed that genotype D was predominant amongst the HBV carriers. Subtype D1 was found in most patients, while two patients were subtype D9. These novel findings may contribute to the body of knowledge and have clinical significance for treating and curing HBV infections in Pakistan.

Circulating miRNA-192 and miR-29a as Disease Progression Biomarkers in Hepatitis C Patients with a Prevalence of HCV Genotype 3.

MicroRNAs miR-29a and miR-192 are involved in inflammatory and fibrotic processes of chronic liver disease, and circulating miR-29a is suggested to diagnose fibrosis progression due to hepatitis C virus (HCV) infection. This study aimed to evaluate the expression profile of circulating miR-192 and 29a in a patient cohort with a high frequency of HCV genotype-3. A total of 222 HCV blood samples were collected and serum were separated. Patients were classified into mild, moderate, and severe liver injury based on their Child-Turcotte-Pugh CTP score. RNA was isolated from the serum and used for quantitative real-time PCR. The HCV genotype-3 (62%) was the predominant HCV genotype. In HCV patients, the serum miR-192 and miR-29a levels were significantly upregulated in comparison to healthy controls (p = 0.0017 and p = 0.0001, respectively). The progression rate of miR-192 and 29a in the patient group with mild was highly upregulated compared to patients with moderate and severe hepatitis infection. The ROC curve of miR-192 and miR-29a of moderate liver disease had a significant diagnostic performance compared to the other HCV-infected groups. The increase in miR-29a and miR-192 serum levels was even slightly higher in patients with HCV genotype-3 than in non-genotype-3 patients. In conclusion, serum miR-192 and miR-29a levels significantly increased during the progression of chronic HCV infection. The marked upregulation in patients with HCV genotype-3 suggests them as potential biomarkers for hepatic disease, independently of the HCV genotype.

Antibiotic Resistance Profiling and Phylogenicity of Uropathogenic Bacteria Isolated from Patients with Urinary Tract Infections.

Urinary tract infections (UTIs) are healthcare problems that commonly involve bacterial and, in some rare instances, fungal or viral infections. The irrational prescription and use of antibiotics in UTI treatment have led to an increase in antibiotic resistance. Urine samples (145) were collected from male and female patients from Lower Dir, Khyber Pakhtunkhwa (KP), Pakistan. Biochemical analyses were carried out to identify uropathogens. Molecular analysis for the identification of 16S ribosomal RNA in samples was performed via Sanger sequencing. Evolutionary linkage was determined using Molecular Evolutionary Genetics Analysis-7 (MEGA-7). The study observed significant growth in 52% of the samples (83/145). Gram-negative bacteria were identified in 85.5% of samples, while Gram-positive bacteria were reported in 14.5%. The UTI prevalence was 67.5% in females and 32.5% in males. The most prevalent uropathogenic bacteria were Klebsiella pneumoniae (39.7%, 33/83), followed by Escherichia coli (27.7%, 23/83), Pseudomonas aeruginosa (10.8%, 9/83), Staphylococcus aureus (9.6%, 8/83), Proteus mirabilis (7.2%, 6/83) and Staphylococcus saprophyticus (4.8%, 4/83). Phylogenetic analysis was performed using the neighbor-joining method, further confirming the relation of the isolates in our study with previously reported uropathogenic isolates. Antibiotic susceptibility tests identified K. pneumonia as being sensitive to imipenem (100%) and fosfomycin (78.7%) and resistant to cefuroxime (100%) and ciprofloxacin (94%). Similarly, E. coli showed high susceptibility to imipenem (100%), fosfomycin (78.2%) and nitrofurantoin (78.2%), and resistance to ciprofloxacin (100%) and cefuroxime (100%). Imipenem was identified as the most effective antibiotic, while cefuroxime and ciprofloxacin were the least. The phylogenetic tree analysis indicated that K. pneumoniae, E. coli, P. aeruginosa, S. aureus and P. mirabilis clustered with each other and the reference sequences, indicating high similarity (based on 16S rRNA sequencing). It can be concluded that genetically varied uropathogenic organisms are commonly present within the KP population. Our findings demonstrate the need to optimize antibiotic use in treating UTIs and the prevention of antibiotic resistance in the KP population.

Field response and molecular screening of European wheat germplasm against powdery mildew at the Himalayan region of Pakistan.

Wheat powdery mildew possesses a significant threat to wheat crops not only on a global scale but also in the northern region of Pakistan. Recognizing the need for effective measures, the exploration and utilization of exotic germplasm take on critical importance. To address this, a series of trials were made to investigate the response of 30 European (EU) lines, in addition to the local checks (Siran, Atta-Habib (AH) and Ghanimat-e-IBGE) against wheat powdery mildew at the Himalayan region of Pakistan. The study involved field testing from 2018 to 2022 across multiple locations, resulting in 38 different environments (location × year). In addition to field evaluations, molecular genotyping was also performed. The disease was absent on the tested lines during 2018, 2019, and 2020 whereas it ranged from 0 to 100% at Chitral location during 2021, where 100% was observed only for one EU wheat line "Matrix." The disease prevailed only at Gilgit location (0-60% for EU wheat line "F236") and at Nagar location (0-10% for EU wheat lines Substance and Nelson) during the disease season of 2022. Most of the EU wheat lines showed very low ACI values, due to an overall low disease pressure. Matrix showed the maximum ACI (1.54) followed by Ritter (1.25) and Bli_autrichion (0.87), whereas the minimum (0.1) was for Substance, JB_Asano, and KWS_Loft followed by Canon (0.19), all exhibiting partial resistance. The molecular marker-based screening revealed that Pm38 was the most prevalent and detected in 100% of wheat lines followed by Pm39 (60%) and Pm8 (30%). Six wheat lines (20%) possessed all three Pm genes (Pm8, Pm38, and Pm39) concurrently. The variability observed in this study can be utilized in future breeding efforts aimed at developing resistant wheat varieties.

Circulating microRNA-122 in HCV cirrhotic patients with high frequency of genotype 3.

MicroRNA-122 (miR-122) is a liver abundant microRNA that is released upon liver injury. In the present study, we investigated the circulating miR-122 profiles in a Pakistani patients´ cohort with HCV chronic liver disease that was mainly based on HCV genotype 3 infections. From 222 patients with chronic HCV liver disease, classified as mild, moderate, or severe, serum samples were collected. Cell-free RNA was isolated and used for miR-122 quantification by qPCR. More than 60% of 222 patients were infected with HCV genotype 3. ALT values and HCV viral load showed no correlation with the HCV genotype. Circulating miR-122 levels were significantly upregulated in patients with cirrhosis. Notably, HCV patients with mild cirrhosis showed the most marked increase in serum miR-122 levels (p = 0.0001). Furthermore, we proved a positive correlation (r = 0.46) of miR-122 with the ALT values in patients with mild cirrhosis. Importantly, our data of increased miR-122 levels in serum samples obtained from a patient cohort with a high prevalence of chronic genotype 3 HCV infection confirmed the previous findings collected from cohorts with a high prevalence of genotype 1. Therefore, we suggest that miR-122 increase after HCV infection does not depend on the HCV genotype. In conclusion, our findings confirm that serum miR-122 levels are significantly upregulated in the HCV cirrhotic patients serving in particular as a biomarker for the non-advanced stages of cirrhosis, independently of the HCV genotype.

Screening of small molecule libraries using combined text mining, ligand- and target-driven based approaches for identification of novel granzyme H inhibitors.

Granzymes are serine proteases synthesized by CTL and NK cells. Five granzyme genes (GzmA, -B, -H, -K, -M) are present in humans, which are located at three different chromosomal loci. Being serine proteases, the binding pocket constitutes a catalytic triad (i.e., His59, Asp103 and Ser197). Granzymes are released into target (cancerous and virally infected) cells by a specialized process known as granule exocytosis pathway. After internalization, these proteases initiate apoptosis. Granzymes are also involved in other non-apoptotic immune associated roles like ECM remodeling, cytokine modulation, killing of pathogens through generation of phagosomes. Their intracellular activity is regulated by specialized inhibitors knows as SERPINs. However, if these proteases are secreted in excess into the extracellular environment, their regulation becomes important as otherwise they start self-damage to the tissues thereby worsening the disease conditions. Efforts are being made to identify potential inhibitors for regulation of these proteases in an extracellular environment. Physiological and synthetic inhibitors have been reported against some members however there is no known inhibitor against extracellular human GzmH. Thus, in the current study, we investigated small molecule databases for the identification of potential molecules having the ability to inhibit GzmH by combined molecular simulations, which can ultimately be used as a potential therapeutic agent.

Phylogenetic analysis of the 5' untranslated region of HCV from cirrhotic patients in Khyber Pakhtunkhwa, Pakistan.

Hepatitis C virus (HCV), a small, single-stranded RNA virus with a 9.6 kb genome, is one of the most common causes of liver diseases. Sequencing of the 5' untranslated region (UTR) is usually used for HCV genotyping, but it is less important in numerous subtypes due to its scarce sequence variations. This study aimed to identify genotypes using the 5' UTR of HCV from cirrhotic patients of Khyber Pakhtunkhwa (KP). Serum RNA samples (44) were screened by real time PCR to determine the HCV viral load. Nested PCR was performed to identify cDNA and the 5' UTR. The HCV 5' UTR was sequenced using the Sanger method. MEGA-7 software was used to analyze evolutionary relatedness. After 5' UTR sequencing, 26 samples (59%) were identified as genotype 3, and 2 samples (6%) were identified as genotypes 1, 2 and 4. The most predominant genotype was 3a, and genotype 4 was rarely reported in the phylogenetic tree. Analysis of the HCV 5' UTR is an efficient alternative method for confirmation of various genotypes. Phylogenetic analysis showed that genotype 3 was dominant in the area of KP, Pakistan.

Screening of FDA approved drugs for finding potential inhibitors against Granzyme B as a potent drug-repurposing target.

Granzyme B is one of the best-characterized and extensively studied member of cytotoxic lymphocytes (CL) proteases. Initially, it is thought to be involved in eliminating virally infected or cancerous cells by using a specialized mechanism through which they are internalized into target cells. In the last decade, however this dimension has changed as there are several reports show that not only CL but also other immune cells can also synthesize Granzyme B. This leads to the possibility of the presence of these proteases in extracellular environment. Being active protease, it then raises the possibility of damaging host tissues as evident from the available reported literature. In many instances, Granzyme B is directly involved in pathogenicity, however in others, it contributes to the disease severity as their over expression makes the clinical situation quite worse which ultimately leads to the chronic state of the disease. Serine protease inhibitor-9 is a natural known intracellular inhibitor of Granzyme B, however there is less data available about the potential inhibitors that can regulate its activity in an extracellular environment. Current study is an effort to identify potential novel inhibitors of Granzyme B. For this aim, drug repurposing study was performed. Around 7900 FDA approved drugs were screened using both ligand- and target-driven approaches. Initially, all molecules were docked using induced fit docking (IFD) approach and selected 318 high-docking scored molecules were used in short (1-ns) molecular dynamics (MD) simulations. Based on MM/GBSA binding free energy calculations, 6 compounds were selected and used in long (100-ns) MD simulations. These compounds were then used in binary QSAR analysis. Therapeutic activity potentials of studied compounds were investigated by Clarivate Analytics's MetaCore/MetaDrug platform which uses binary QSAR models. It is developed based on manually curated database of molecular interactions, molecular pathways, gene-disease associations, chemical metabolism and toxicity information. Results of selected compounds were compared with a positive control molecule. Current drug repurposing study is a step ahead in finding potential lead compounds by screening database of FDA approved molecules. We have identified novel inhibitors (Tannic acid, Mupirocin, Phytonadiol sodium diphosphate, Cefpiramide, Xenazoic acid) that have potential to decrease the activity of Granzyme B.

The Resurgence of Measles Infection and its Associated Complications in Early Childhood at a Tertiary Care Hospital in Peshawar, Pakistan.

Measles infection is of substantial interest to immunologists due to its paradoxical interaction with the immune system. After the acquisition of the measles infection, secondary infection plays a pivotal role in measles-related deaths. A cross-sectional study conducted between December 2018 and July 2019 is presented here. A total of one hundred children of both genders presented with measles complications were included following WHO criteria. Measles confirmation was done by quantitative determination of anti-measles antibodies (IgM) in patients' sera while patient-related demographic data, vaccination status, and other clinical information were obtained on a separate form. The number of female patients (52%) slightly exceeded the number of males (48%). 43% of patients' parents were illiterate, and half of the patients (50%) were from a poor background. The majority of children (76%) who presented with the complications did not receive a measles vaccine. 56% of children were breastfed while 58% received vitamin A supplements but developed complications. The elevated levels of anti-measles IgM were observed in 77% of cases. In both genders, the major complications were pneumonia, lower respiratory tract infection (LRTI), acute diarrhea, diarrhea and LRTI, pneumonia and diarrhea, otitis media and pneumonia, myocarditis and LRTI, and pneumothorax. The majority of the infected children (n = 48) under 12 months of age had associated complications. It has been observed that the measles virus strikes early age children in the northwestern region of Pakistan, which is an alarming situation and is associated with the aforementioned complications, especially in unvaccinated children. Anti-measles IgM is an important serological parameter for early diagnosis of measles infection.Measles infection is of substantial interest to immunologists due to its paradoxical interaction with the immune system. After the acquisition of the measles infection, secondary infection plays a pivotal role in measles-related deaths. A cross-sectional study conducted between December 2018 and July 2019 is presented here. A total of one hundred children of both genders presented with measles complications were included following WHO criteria. Measles confirmation was done by quantitative determination of anti-measles antibodies (IgM) in patients' sera while patient-related demographic data, vaccination status, and other clinical information were obtained on a separate form. The number of female patients (52%) slightly exceeded the number of males (48%). 43% of patients' parents were illiterate, and half of the patients (50%) were from a poor background. The majority of children (76%) who presented with the complications did not receive a measles vaccine. 56% of children were breastfed while 58% received vitamin A supplements but developed complications. The elevated levels of anti-measles IgM were observed in 77% of cases. In both genders, the major complications were pneumonia, lower respiratory tract infection (LRTI), acute diarrhea, diarrhea and LRTI, pneumonia and diarrhea, otitis media and pneumonia, myocarditis and LRTI, and pneumothorax. The majority of the infected children (n = 48) under 12 months of age had associated complications. It has been observed that the measles virus strikes early age children in the northwestern region of Pakistan, which is an alarming situation and is associated with the aforementioned complications, especially in unvaccinated children. Anti-measles IgM is an important serological parameter for early diagnosis of measles infection.

Potent novel inhibitors against hepatitis C virus NS3 (HCV NS3 GT-3a) protease domain.

HCV NS3, a non-structural hepatitis C viral protein is used as one of the potential targets for inhibition by direct-acting antivirals. It is known that the success rate for HCV genotype-1 treatment remained very high, however, treatment of genotype-3a (GT-3a), is still quite challenging. In the current study, the HCV GT-3a full-length NS3 gene was amplified and sequenced. The complete nucleotide sequence was translated into the amino acid sequence and homology models of HCV-NS3 GT-3a were generated by HCV-NS3 genotype-1b as a template. The objective of the study was to screen novel therapeutic hits from large databases. For this aim, various small molecule databases including, BindingDB (∼45.000 compounds), NCI (∼265.000 compounds), and Specs-SC (∼212.000 compounds) were used. Firstly, all of the compounds were screened using binary-QSAR models from the MetaCore/MetaDrug server, and compounds were filtered based on therapeutic activity predictions by the anti-viral QSAR model. Filtered molecules were used in 26 different toxicity QSAR models and active non-toxic compounds were identified. These selected molecules were then used in docking and molecular dynamics (MD) simulations studies at the binding cavities of the NS3 protease domain of the GT-3a. Results were compared with known inhibitors and novel molecules are proposed against HCV-NS3 GT-3a. These molecules have high ligand efficiencies as compared to the reference molecules suggesting a better alternate to the existing suite of inhibitors. Thus, this study will be a step ahead in the development of new potential compounds as antiviral drugs for the GT-3a target.